In lymphocytes and mast cells, stimulation through antigen or Fc receptors leads to depletion of intracellular Ca2+ stores and subsequent Ca2+ influx through specialized Ca2+-release-activated Ca2+ (CRAC) channels in the plasma membrane. The increase in intracellular free Ca2+ ([Ca2+],) levels activate the phosphatase calcineurin, the upstream regulator of the transcription factor NFAT. We describe a strategy, based on global RNAi screening in Drosophila, to identify genes whose products influence [Ca2+ levels, calcineurin activation or localization of NFAT. RNAi in Drosophila cells is much more efficient than in mammalian cells, providing a powerful method to test individually the effects of specific disruption of all genes in the Drosophila genome. We have completed the first phase of this screen, identifying dsRNAs that cause nuclear localization of an NFAT-GFP fusion protein in resting Drosophila cells and so normally function to retain NFAT in the cytoplasm, for instance by maintaining [Ca2+]i and calcineurin activity at low levels in resting cells or counteracting the nuclear shuttling of NFAT. In Aim 1, we will continue analyzing the regulators identified in this screen. In Aim 2, we will perform a new screen to identify proteins that increase [Ca2+]| levels and calcineurin activity or promote the nuclear translocation of NFAT, by screening for dsRNAs that prevent NFAT nuclear import in Drosophila cells stimulated with thapsigargin which causes depletion of Ca2+ stores. In Aim 3, we will assess the involvement of specific candidates previously implicated in Ca2+-calcineurin-NFAT signalling, which may not have shown up in the screens because of redundancy or effects on cell viability or cytoskeletal function. Once regulators that function in Drosophila have been validated, we will test the functions of the mammalian homologues. These studies are expected to provide a comprehensive picture of the signalling networks that modulate [Ca2+]j levels, calcineurin activity and NFAT subcellular localization in Drosophila and mammalian cells. The studies are important because the Ca2+-calcineurin-NFAT signalling pathway has a critical role in many developmental and transcriptional programs, including T cell activation and differentiation, osteoclast differentiation, cardiac valve development, slow-twitch skeletal muscle fiber differentiation, and neuronal and vascular patterning. NFAT is also implicated in many pathological processes, including transplant rejection, inflammation, osteoporosis, myocardial hypertrophy, allergy and autoimmune disease.